Recent studies have revealed unconventional myosin V to be an important actin-based molecular motor involved in vesicular movement. We have cloned the cDNA for Drosophila myosin V and have shown that its heavy chain is encoded by a single gene at 43C-D. A fly line was identified in which a P[lacW] transposon was inserted just upstream from the coding region of the myosin V gene. The phenotype is recessive embryonic lethal and probably represents a hypomorph. We are in the process of hopping out the P-element in order to create a fly for transgene experiments. To this end, we are constructing a vector containing the entire myosin V gene. This system will be used to express a GFP-tagged wild-type myosin V gene and various myosin V mutations to test the function of portions of the molecule. In separate experiments, we have expressed the neck region of Drosophila myosin V as a GST-fusion protein. The neck region consists of six light chain binding sites (termed IQ motifs). Using a blot overlay method, we have probed the binding of calmodulin and essential light to this. Both calmodulin and essential light chain are able to bind to the six-IQ neck region. We next truncated the neck region by removal of the first IQ motif. It is possible that essential light chain binds to this first IQ motif, since the essential light chain binds to the first IQ motif in myosin II molecules. This five IQ motif still binds both calmodulin and essential light chain suggesting the essential light chain binding site resides in another IQ motif. Finally, we are initiating studies on a novel Drosophila myosin that was uncovered by the Drosophila genome project. The entire genomic DNA for this myosin has been deposited into the gene bank. Analysis of the sequence in comparison to other myosins has allowed us to make a guess of the coding sequence for this myosin. Analysis of this sequence suggests that this may be a novel myosin superfamily member. We are currently isolating cDNA clones for the myosin and will be examining Drosophila lines known to carry various mutations in this region.